Methods and apparatus for assays of bacterial spores

ABSTRACT

A sample of unknown bacterial spores is added to a test strip. The sample of unknown bacterial spores is drawn to a first sample region on the test strip by capillary action. Species-specific antibodies are bound to the sample when the unknown bacterial spores match the species-specific antibodies, otherwise the sample is left unbound. DPA is released from the bacterial spores in the bound sample. The terbium ions are combined with the DPA to form a Tb-DPA complex. The combined terbium ions and DPA are excited to generate a luminescence characteristic of the combined terbium ions and DPA to detect the bacterial spores. A live/dead assay is performed by a release of the DPA for live spores and a release of DPA for all spores. The detection concentrations are compared to determine the fraction of live spores. Lifetime-gated measurements of bacterial spores to eliminate any fluorescence background from organic chromophores comprise labeling the bacterial spore contents with a long-lifetime lumophore and detecting the luminescence after a waiting period. Unattended monitoring of bacterial spores in the air comprises the steps of collecting bacterial spores carried in the air and repeatedly performing the Tb-DPA detection steps above. The invention is also apparatus for performing the various methods disclosed above.

RELATED APPLICATIONS

[0001] The present application is related to U.S. Provisional PatentApplication serial No. 60/353,268 filed on Feb. 1, 2002; U.S.Provisional Patent Application serial No. 60/395,372, filed on Feb. 1,2002; U.S. Provisional Patent Application serial No. 60/414,170, filedon Sep. 27, 2002, and U.S. Provisional Patent Application serial No.(CIT3770), filed on ______, each of which is incorporated herein byreference and to which priority is claimed pursuant to 35 USC 119.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention is directed assays of bacterial endospore levels.

[0004] 2. Description of the Prior Art

[0005] The prior art for species-specific bacterial spore detection,using the lateral flow immunoassay method, is based on observing the redcolor of gold nanoparticles. It uses two antibodies, in combination, tospecifically detect the bacterial spore species of interest in solution.One of the antibodies is attached to a colloidal gold nanoparticle, andthe other antibody is immobilized on the nitrocellulose membranedownstream from the point of sample introduction. When about 100 μl ofsample is added to the test strip membrane on top of the area 30 thatcontains the colloidal gold labeled antibodies, specific binding betweenbacterial spores and gold labeled antibodies occurs. Simultaneously,capillary action moves the gold labeled antibodies (both spore bound andnot bound) along the strip membrane 32. In the sample region 34 of thetest strip 32 (downstream), specific binding of a second antibodycaptures bacterial spores with the attached colloidal gold labeledantibody, which gives rise to a red line in the sample region 34 due tothe immobilized gold nanoparticles as shown in the bottom left ofFIG. 1. In the control region 36 of the test strip 32 (furtherdownstream), as an internal control, a polyclonal antibody binds thegold labeled antibodies that did not bind bacterial spores of interest,which also gives rise to a red line. Thus, observation of two bands, oneeach in the sample and control regions, indicates a positive test forthe bacterial spore of interest. The observation of only one band asshown in the bottom right of FIG. 1 is a negative test result. Thefundamental limitation of this method is its sensitivity; a minimumconcentration of 10⁵ spores/ml is needed before the red color from thegold nanoparticles becomes detectable; for reference, a 100 μl samplecontaining 10,000 anthrax spores is lethal.

[0006] Therefore what is needed is a method for improving the detectionlimit of lateral flow immunoassay based detection of bacterial spores,which is reported to be 10⁵ spores/ml. This prior art detection limitprevents detection of trace quantities of bacterial spores. A tracequantity of 8000 anthrax spores, for example, is enough fill a person.

[0007] The prior art the method for determining the fraction of viablebacterial spores is based on two measurements. First, the viablebacterial spore count is measured by colony counting, and second, thetotal bacterial spore count is measured by direct microscopic counting.The ratio of viable to total bacterial spore count yields the fractionof spores that remain viable within a given sample. The procedure forcolony counting to determine endospore concentration is comprised of thesteps of (1) heat shocking the sample to kill vegetative cells whilebacterial spores remain viable, (2) plating a known volume of the samplewith a known dilution factor onto a growth medium, and (3) incubatingthe growth plates for 2 days. Finally, the resulting visible coloniesare counted and reported as colony forming units (CFU's). The procedurefor direct microscopic counting is comprised of the steps of (1) placingthe sample on a slide with an indentation of a known volume. The glasssurface of the slide is inscribed with squares of known area. (2) Thebacteria in each of the several squares are counted and the averagecount is multiplied by an appropriate factor to yield the number oftotal cells per milliliter in the original suspension.

[0008] These methods suffer prohibitive difficulties with lowconcentration samples collected in the field. First, bacterial sporestend to attach themselves onto particulates (dust etc.) and may easilyrepresent the bulk of the biomass in a field sample. Unfortunately,attached bacterial spores cannot be counted with either colony countingor direct microscopic counting. Second, colony-counting methods onlywork for cultivable bacteria, which are in the minority in field samples(<10% of microbial species form colonies). Finally, the traditionalmethods are lengthy (>2 days) and labor intensive. These problems havemade quantification of low concentration field samples extremelydifficult, and have subsequently prevented the application of thesemethods towards a reliable and/or real-time bacterial spore live/deadassay.

[0009] There is a need to develop a live/dead assay for bacterialspores, because there is a need to measure the fraction of bacterialspores that remain viable for samples exposed to harsh environmentalconditions such as desert and arctic environments. In terms of planetaryprotection, which is primarily concerned with spacecraft sterilization,in order to improve sterilization procedures, one must measure thefraction of viable spores after completion of various sterilizationprotocols. The samples of interest contain low bacterial sporeconcentration and many particulates, for which the prior art methodsuseless.

[0010] Prior art methods for monitoring aerosolized bacterial sporesincludes air filtering with subsequent PCR analysis of gene segmentsfrom species of interest, and aerosol sampling with subsequent culturingand colony counting. The PCR based method is strongly dependent onimpurities in the air, such as city pollution, and requires speciallytrained technicians to perform sample preparation prior to running thePCR reaction. The procedure for colony counting, which is comprised of(1) heat shocking the sample to kill vegetative cells while bacterialspores remain viable, (2) plating a known volume of the sample with aknown dilution factor onto a growth medium, (3) incubating the growthplates for two days, also requires the active participation of atechnician. This also assumes that the spore forming microbes arecultivable. It is estimated that only 10% of bacterial species arecultivable. The cost of labor, technical complexity of PCR and slowresponse time of colony counting have prevented the wide spreadapplication of these methods for monitoring of bacterial spores in theair.

BRIEF SUMMARY OF THE INVENTION

[0011] Lateral Flow Immunoassay

[0012] One embodiment of the invention is defined as a method forlateral flow immunoassay for bacterial spore detection andquantification comprising the steps of providing a matrix includingterbium ions, releasing DPA from the bacterial spores, combining theterbium ions with the DPA in solution, and exciting the combined terbiumions and DPA to generate a luminescence characteristic of the combinedterbium ions and DPA to detect the bacterial spores. The matrix Thedetection of the spores or their concentration above a predeterminedthreshold generates an alarm signal.

[0013] The DPA is released from the bacterial spores by microwaving thespores, germinating the spores with L-alanine, sonicating the sporeswith microspheres or autoclaving the spores. These methods by no meansnecessarily exhaust the ways in which the DPA can be released from thespores and all other methods of lysing the spores are deemed equivalent.

[0014] Exciting the combined terbium ions and DPA generates aluminescence characteristic of the combined terbium ions and DPA. Thisis achieved by radiating the combined terbium ions and DPA withultraviolet light. Again, any method by which luminescence can beinduced is included within the scope of the invention and is deemed tobe equivalent.

[0015] The invention can also be characterized as a method for lateralflow immunoassay for bacterial spore detection and quantification. Themethod starts with the step of adding a sample of unknown bacterialspores to a test strip. The sample of unknown bacterial spores is drawnto a first sample region on the test strip by capillary action.Species-specific antibodies are bound to the sample when the unknownbacterial spores match the species-specific antibodies, otherwise thesample is left unbound. DPA is released from the bacterial spores in thebound sample. The terbium ions are combined with the DPA to form aTb-DPA complex. The combined terbium ions and DPA are excited togenerate a luminescence characteristic of the combined terbium ions andDPA to detect the bacterial spores.

[0016] The method further comprises the steps of performing the samesteps with a standard of known bacterial spores with knownconcentration. The sample is added to the test strip and drawn to asecond sample region on the test strip. Species-specific antibodies areselectively bound to the standard when the known bacterial spores matchthe species-specific antibodies, otherwise the standard unbound is leftunbound. DPA is released from the bacterial spores in the bound standardand combined with the terbium ions. The combined terbium ions and DPAare excited to generate a luminescence characteristic of the combinedterbium ions and DPA to detect the bacterial spores of the standard. Theintensity of the excited luminescence from the sample is compared withthe excited luminescence from the standard to derive a quantification ofthe spore concentration in the sample. The method may further comprisethe step of confirming arrival of the sample and standard in the firstand second sample regions respectively by means of a visual indicator.

[0017] Live/Dead Assay for Bacterial Spores

[0018] The invention is defined in another embodiment as a method forlive/dead assay for bacterial spores comprising the steps of: providinga solution including terbium ions in a sample of live and dead bacterialspores; releasing DPA from viable bacterial spores by germination from afirst unit of the sample; combining the terbium ions with the DPA insolution released from viable bacterial spores; exciting the combinedterbium ions and DPA released from viable bacterial spores to generate afirst luminescence characteristic of the combined terbium ions and DPAto detect the viable bacterial spores; releasing DPA from dead bacterialspores in a second unit of the sample by autoclaving, sonication ormicrowaving; combining the terbium ions with the DPA in solutionreleased from dead bacterial spores; exciting the combined terbium ionsand DPA released from dead bacterial spores to generate a secondluminescence characteristic of the combined terbium ions and DPA todetect the dead bacterial spores; generating a ratio of the first tosecond luminescence to yield a fraction of bacterial spores which arealive.

[0019] Lifetime-Gated Measurements of Bacterial Spores and ImagingBacterial Spores Using an Active Pixel Sensor

[0020] In yet another embodiment the invention is a method forlifetime-gated measurements of bacterial spores to eliminate anyfluorescence background from organic chromophores comprising the stepsof providing a solution including terbium ions with a sample ofbacterial spores; labeling the bacterial spore contents with along-lifetime lumophore; releasing DPA from the bacterial spores;combining the terbium ions with the DPA in solution; exciting thecombined terbium ions and DPA for a first time period; waiting a secondtime period before detecting luminescence; and detecting a luminescencecharacteristic of the combined terbium ions and DPA after the secondtime period during a defined temporal window synchronized withluminescence timed from the long lifetime lumophore to detect thebacterial spores.

[0021] In one embodiment the first time period of excitation is of theorder of nanoseconds, the second time period is of the order ofmicroseconds and the defined temporal window is of the order ofmilliseconds.

[0022] In another embodiment the first time period of excitation is ofthe order of 1-10 nanoseconds, where the second time period is of theorder of tens of microseconds and where the defined temporal window isof the order of 1-10 milliseconds.

[0023] In still another embodiment the first time period of excitationis of the order of nanoseconds, the second time period is of the orderof tenths to tens of milliseconds and where the defined temporal windowis of the order of hundreds of microseconds.

[0024] Unattended Monitoring of Bacterial Spores in the Air

[0025] In yet another embodiment the invention is a method forunattended monitoring of bacterial spores in the air comprising thesteps of collecting bacterial spores carried in the air; suspending thecollected bacterial spores in a solution including terbium ions;releasing DPA from the bacterial spores; combining the terbium ions withthe DPA in solution; exciting the combined terbium ions and DPA togenerate a luminescence characteristic of the combined terbium ions andDPA; detecting the luminescence to determine the presence of thebacterial spores; and generating an alarm signal when the presence ofbacterial spores is detected or the concentration thereof reaches apredetermined magnitude.

[0026] The step of collecting bacterial spores carried in the aircomprises capturing the bacterial spores with an aerosol sampler orimpactor. The step of detecting the luminescence to determine thepresence of the bacterial spores comprises monitoring the luminescencewith a spectrometer or fluorimeter.

[0027] Preferably, the step of collecting bacterial spores carried inthe air comprises continuously sampling the air and the step ofdetecting the luminescence to determine the presence of the bacterialspores comprises continuously monitoring the luminescence.

[0028] When the step of releasing DPA from the bacterial sporescomprises microwaving the bacterial spores to heat the solution, thestep of combining the terbium ions with the DPA in solution comprisescooling the heated solution to increase the fraction of bound Tb-DPAcomplex.

[0029] The invention is also apparatus for performing the variousmethods disclosed above. For example, the invention includes anapparatus for unattended monitoring of bacterial spores in the aircomprising: a biosampler for capturing the bacterial spores in the airand having a collection vessel containing a solution including terbiumions into which the captured bacterial spores are suspended; means forreleasing DPA from the bacterial spores in the solution to allow the DPAto combine with the terbium ions to form a Tb-DPA complex; an energysource for exciting the Tb-DPA complex to generate luminescence; anelectro-optical circuit to measure the luminescence; and an alarmcircuit coupled to the electro-optical circuit to detect a bacterialspore concentration above a predetermined threshold.

[0030] The invention is also an apparatus for lateral flow immunoassayfor bacterial spore detection and quantification comprising: a strip ofmaterial for providing lateral capillary flow of a solution includingterbium ions across the strip; an input region on the strip forreceiving a liquid sample containing terbium ions, the first zone beingprovided with a first antibody for specific binding to a specific specieof bacterial spores; a sample region of the strip laterally displacedfrom the input region and communicated thereto by means of capillaryflow therebetween, the sample region being provided with a secondantibody to capture bacterial spores with the attached first antibodyand to immobilize them; means for releasing DPA from the bacterialspores in the sample region of the strip to then allow the terbium ionsto combine with the DPA in solution; an energy source to excite thecombined terbium ions and DPA in the sample region of the strip togenerate a luminescence characteristic of the combined terbium ions andDPA; and a luminescence detector to identify the presence or measure theconcentration of the bacterial spores in the sample region of the strip.

[0031] While the apparatus and method has or will be described for thesake of grammatical fluidity with functional explanations, it is to beexpressly understood that the claims, unless expressly formulated under35 USC 112, are not to be construed as necessarily limited in any way bythe construction of “means” or “steps” limitations, but are to beaccorded the full scope of the meaning and equivalents of the definitionprovided by the claims under the judicial doctrine of equivalents, andin the case where the claims are expressly formulated under 35 USC 112are to be accorded full statutory equivalents under 35 USC 112. Theinvention can be better visualized by turning now to the followingdrawings wherein like elements are referenced by like numerals.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 is a diagram of a prior art lateral flow immunoassay forbacterial spores.

[0033]FIG. 2a is a microscopic image of a spore (about 1 μm in diameter)highlighting a DPA rich spore core.

[0034]FIG. 2b is a diagram of a Tb³⁺ ion (shaded ball) by itself has alow absorption cross section (<10 M⁻¹cm⁻¹) and consequently has lowluminescence intensity. The Tb³⁺ ion can bind the light harvesting DPA(absorption cross section>10⁴ M⁻¹) originating from the spore; DPAbinding gives rise to bright Tb luminescence.

[0035]FIG. 2c is a diagram of a photophysical scheme for DPA sensitizedluminescence of the Tb complex (absorption-energy transfer-emission,AETE).

[0036]FIG. 3 is a diagrammatic illustration showing a few drops ofbacterial spore containing sample are added to the test strip membrane.

[0037]FIGS. 4a-4 c are graphs of the intensity of Tb luminescence versestime. FIG. 4a shows the intensity during germination starting with t=0when L-alanine was added. FIG. 4b shows the Tb luminescence aftercompletion of germination corresponding to Tb-DPA complex. FIG. 4c showsTb luminescence induced by autoclaving.

[0038]FIG. 5 is a diagram illustrating the active pixel sensor imagingmethod as applied to Tb luminescence in bacterial spores.

[0039]FIG. 6 is the lifetime series decay of the bacterial sporesilluminated in FIG. 5.

[0040]FIG. 7 is a simplified diagram of an unattended air monitor forbacillus using Tb-DPA detection.

[0041]FIGS. 8a and 8 b are graphs of the relative luminescence intensityas a function of time and wavelength respectively. FIG. 8a illustratesthe time course of spore monitoring and FIG. 8b shows the spectrum justbefore spore release, less than 15 minutes after spore release and 60minutes after spore release.

[0042] The invention and its various embodiments can now be betterunderstood by turning to the following detailed description of thepreferred embodiments which are presented as illustrated examples of theinvention defined in the claims. It is expressly understood that theinvention as defined by the claims may be broader than the illustratedembodiments described below.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Lateral Flow Immunoassay

[0044] The invention is directed to lateral flow immunoassay forbacterial spore detection and quantification using lanthanideluminescence with both high sensitivity and selectivity in less thanfive minutes. The method combines lateral flow immunoassay anddipicolinic acid (DPA) triggered terbium (Tb) luminescence technologies.The lateral flow immunoassay provides high selectivity for specificbacterial spore species, and the DPA triggered Tb luminescence methodfor bacterial spore detection enables greatly improved detection limitsover the prior art detection schemes.

[0045] The new technology has significantly improved detection limits,because it is based on Luminescence turn-on against a dark background,which is much more sensitive than measuring the scattered light, fromgold nanoparticles against a bright background. Based on DPA-triggeredTb luminescence experiments, we anticipate single spore detection limitsfor 100 μl samples (i.e. 10 spores/ml).

[0046] The solution for developing a lateral flow immunoassay baseddetection of bacterial spores with single spore detection limits is touse DPA triggered Tb luminescence as the detection scheme. Themethodology for achieving single spore detection is more expresslydisclosed in copending U.S. patent application entitled “An ImprovementIn A Method For Bacterial Endospore Quantification Using LanthanideDipicolinate Luminescence,” Ser. No. (CIT3405) ______, filed ______, andassigned to the same assignee as the present invention, whichapplication is incorporated herein by reference.

[0047] Consider now the DPA-triggered Tb luminescence detection ofbacterial spores. Dipicolinic acid DPA, 2,6 pyridinedicarboxylic acid)is present in high concentrations (about 1 molar or about 15% of byweight) in the core of bacterial spores 38 as a 1:1 complex with Ca²⁺ asshown in FIG. 2a. For all known lifeforms, DPA is unique to bacterialspores and is released into bulk solution upon germination, which is theprocess of spore-to-vegetative cell transformation. Thus, DPA is anindicator molecule for the presence of bacterial spores. Fortuitously,DPA is also a classic inorganic chemistry ligand that binds metal ionswith high affinity. DPA binding to terbium ions triggers intense greenluminescence under UV excitation as shown in FIGS. 2b and 2 c. Thus, thegreen luminescence turn-on signals the presence of bacterial spores, andthe intensity of the luminescence can be correlated to the number ofbacterial spores per milliliter. Potential interferents such as sugars,nucleic and amino acids are present in much lower concentrations inendospores and vegetative cells and have binding constants for Tb thatare approximately six orders of magnitude less than that of DPA(K_(A)=10^(8.7) M⁻¹). This method is relatively immune to theseinterferents.

[0048] The core of bacterial spores contains 1 molar dipicolinic acid(DPA) (˜15% of the spore dry weight). It has been shown that the DPA canbe released into bulk solution by microwaving the sample (germinationwith L-alanine, sonication with microspheres, and autoclaving have alsobeen used to release DPA from the spore). When the released DPA bindsterbium ions in bulk solution, bright green luminescence is triggeredunder UV excitation.

[0049] The mechanism of DPA-triggered Tb luminescence is based on theunique photophysical properties of lanthanide ions. The luminescence oflanthanide ions is characterized by long lifetimes (0.1 to 1 ms), smallextinction coefficients (a.k.a. absorbtivity, about 1 M⁻¹ cm⁻¹) andnarrow emission bands. These characteristics arise because the valence forbitals are shielded from the environment by the outer 5 s and 5 pelectrons, and because the transition between the emitting excited stareand ground state is highly forbidden. Thus, direct excitation of terbiumions leads to weak luminescence due to the small absorption crosssection. However, coordination of aromatic chromophores, like DPA,triggers intense terbium luminescence. The juxtaposition of DPA, whichhas an absorbtivity of 5000 M⁻¹ cm⁻¹ serves as a light-harvesting center(e.g. antenna effect). Strong electronic coupling and downhill energiesallow the DPA centered excitation energy to be efficiently transferredto the lanthanide ion, which subsequently luminesces bright green.

[0050] Consider now the details of lateral flow immunoassay withDPA-triggered Tb luminescence detection of bacterial spores 10. The teststrip 18 is comprised of a nitrocellulose membrane 12 that hasspecies-specific antibodies bound in the sample regions, which areregions 26 and 22 of the strip as shown in FIG. 3. Region 26 containsantibodies for the bacterial spore species 10 of interest (e.g. B.anthracis antibody), and region 22 contains antibodies for B. subtilis(standard 20). First, about 100 μl of sample 10 in a liquid, such aswater, and standard 20 in a solution of the same or a different liquidare added to their respective test strip membranes 12 and 16 in thesample port region 30. Capillary action moves the spores 10 along thestrip membrane 12 and 16. In the sample region 14 of the test strip 12(downstream), specific binding of membrane-bound antibodies captures andimmobilizes the bacterial spores 10, while components of the sample 10that do not bind the antibody continue to flow out of the sample region14. Regions 24 contain an indicator, such as cobalt chloride, thatchanges visible color when the liquid or solvent front arrives, afterabout five minutes, which should suffice to provide adequate separationof the specific binding components to the nonspecific components of thesample 10. For example, where the indicator is cobalt chloride, thecolor changes to blue to pink on arrival of the liquid, which is in thisembodiment is water. The choice of liquid and indicator is a matter ofdesign choice and many other selections can be equivalently substituted.

[0051] In the next step, DPA is released from the core of the spores 10by microwaving the test strip 12. The released DPA binds Tb dissolved inthe solution and triggers green luminescence, which signals the presenceof bacterial spores. The green luminescence can be read or measured by aconventional spectrometer or fluorometer (not shown).

[0052] The control is performed on a parallel test strip to which about100 μl containing a known concentration of Bacillus subtilis is added.The standard 20 undergoes the identical procedure as the unknown sample10. Green luminescence in region 22 and a change in color in regions 24indicates that the assay has worked properly and the ratio ofluminescence intensity from the sample 10 in region 26 and standard 20in region 22 is proportional to the concentration of the bacterial sporeof interest. The microwaving step can be completed in less than 2minutes. Thus the complete assay can be performed within 7 to 10minutes. The sample 10 and standard 20 may be processed simultaneouslyor sequentially as may be desired.

[0053] Live/Dead Assay for Bacterial Spores

[0054] The invention also includes a method and apparatus to measure thefraction of bacterial spores that remain viable or alive, hence alive/dead assay for bacterial spores. The method combines dipicolinicacid triggered terbium luminescence and dipicolinic acid release from(1) viable bacterial spore through germination, and (2) all viable andnonviable bacterial spores by autoclaving, sonication, or microwaving.The ratio of the results from steps (1) and (2) yield the fraction ofbacterial spores that are alive.

[0055] The invention does not suffer from the aforementioned prior artproblems of colony or microscopic counting, because it is based on amolecular approach that (1) works whether or not a bacterial spore isattached on a particulate, (2) does not require bacteria to becultivable, and (3) can be performed on the timescale of 20 minutes.

[0056] The solution for developing a live/dead assay for bacterialspores requires a molecular approach. DPA can be released into bulksolution by inducing germination with L-alanine or by autoclaving thesample. In germination, only viable spores release DPA, whileautoclaving forces all spores, viable and nonviable, to release DPA.Microwaving and sonication also releases DPA from all spores, whetherdead or alive. Again, when the released DPA binds terbium ions in bulksolution, bright green luminescence is triggered under UV excitation.

[0057] The luminescence intensity can be correlated to the concentrationof viable bacterial spores when germination is used to release the DPA,and to the total bacterial spore concentration when either autoclaving,sonication, or microwaving is used to release DPA. Thus, these methodsof DPA release allow us to quantify both the viable and total bacterialspore count, and subsequently the fraction of spores that are viable fora given sample.

[0058] Since germination releases the DPA content of viable bacterialspores, while autoclaving, sonication, and microwaving releases the DPAcontent of all bacterial spores, including non-viable bacterial spores,using the DPA triggered Tb luminescence method in conjunction with theDPA release, induced by (1) germination and (2) either autoclaving,sonication, and microwaving, allows us to determine the viable and totalspore count, respectively, and subsequently the fraction of viablebacterial spores as illustrated in FIGS. 4a, 4 b and 4 c. FIG. 4a showsthe time course data of endospore germination monitored by DPA triggeredTb luminescence at 543 nm. Time zero corresponds to L-alanine inducedgermination. FIG. 4b shows the spectrum of the luminescencecorresponding to Tb-DPA complex, which is induced after completion ofgermination. FIG. 4c compares the spectrum for an autoclaved sampleverses a control sample which is not autoclaved.

[0059] Lifetime-Gated Measurements of Bacterial Spores and ImagingBacterial Spores Using an Active Pixel Sensor

[0060] Finally, the method of the invention is amenable tolifetime-gated measurements to eliminate any fluorescence backgroundfrom organic chromophores. It is also possible to quantify the fractionof bacterial spores that remain viable by inducing DPA release bygermination and microwaving as described below, and to obtain furtherincreased sensitivity by preparing special Tb complexes that enhance theluminescence turn-on, and DPA binding affinity.

[0061] Consider now the problem of imaging bacterial spores. The imagingmethodology is again based on a combination of dipicolinic acidtriggered terbium luminescence (Tb luminescence assay) and imaging usingan active pixel sensor (APS), which is well known to the art. The Tbluminescence assay enables specific detection of bacterial spores with acurrent detection limit of 5,000 spores/ml when TbCl₃ is used as theanalysis reagent. This assay can be performed in 30 minutes or lessdepending on the DPA release mechanism that is employed. APS is ideallysuited to image the resultant Tb luminescence when spores are presentbecause of its inherent ability to perform lifetime gated imaging.

[0062] In this embodiment the spores or their contents have been labeledwith a long-lifetime lumophore which fact is used to advantage duringdetection. Since almost every natural fluorescent material decays in afew nanoseconds, delayed luminescence is a powerful discriminatoragainst background biological or mineralogical signals. For example,flavinoids, NADH, collagen and many other biological and cellularcomponents fluoresce in the wavelength region of 300-500 nm, but allhave lifetimes less than a few tens of nanoseconds.

[0063] Jet Propulsion Laboratory has developed a true snapshot imager,using CMOS technology in an APS that is ideally suited for imaging andmeasurement of delayed luminescence probes. In this implementation, theentire imager can be cycled off and on in a clock cycle, typically lessthan a microsecond. The basic measurement cycle is to pulse anexcitation source for the luminescence with an on time of a fewnanoseconds, wait 30 μs and than turn on the imager for 2 ms, turn itoff and read out the image and the photon counts for each pixel. Aunique feature of the CMOS or Active Pixel Sensor (APS) technology isthat each pixel can contain active circuit elements and can performsignal averaging to improve the signal to noise as well as otherprocessing. By imaging the collection tape, we can count the pixels thatcontain luminescence signal and get a spore count.

[0064]FIG. 5a shows a diagrammatic timing sequence for excitation, adelay φ, and detector integration time Δ. Image data taken with the APSfor an Europium probe with a lifetime of ˜8O0 μs is shown in FIG. 5b inwhich we applied a few spots of the Europium probe to an APS 256×256imager and excited the fluorescence with a pulsed N₂ laser at 337 nm anda pulse width of ˜4 ns. Excitation can be performed with a compactlaser, laser diode or LED. By adjusting the timing of the detectionwindow, delay φ, the decay curve of the fluorophore can be mapped out asshown in FIG. 6, which is a graph of the lifetime data obtained from theimages of FIG. 5b. The fluorescence signal in FIG. 6 is summed up fromall the pixels on the upper spot of the APS sensor as shown in FIG. 5b.

[0065] Unattended Monitoring of Bacterial Spores in the Air

[0066] Consider now the technology that is required to enable one toachieve unattended monitoring of bacterial spores in the air. Thenovelty of the method lies again in the combination of (1) aerosolcapture methods and (2) lanthanide luminescence detection of bacterialspores This combination will enable an alarm for airborne bacterialspores similar in concept to a smoke detector, which works continuouslyand unattended.

[0067] The invention as described below does not suffer from the abovementioned problems of the prior art, because it (1) does not requirecultivable bacteria, and (2) can be performed continuously with asampling rate of at least four readings per hour using currentinstrumentation, and (3) does not require active sampling by a trainedtechnician.

[0068] Online monitoring of aerosolized bacterial spores, such asBacillus anthracis and Clostridium botulism spores, is essential inlocations such as public transportation, mail sorting, food preparation,health care facilities and even military environments. We have becomeespecially motivated to develop a method of unattended monitoring ofbacterial spores in the air after the anthrax attacks following the Sep.11, 2002 terrorist attacks. Another motivation was the application ofthe method towards planetary protection, which is primarily concernedwith spacecraft sterilization.

[0069] A solution for unattended monitoring of airborne bacterial sporesis achieved by the combination of (1) aerosol capture methods and (2)lanthanide luminescence detection of the bacterial spores as describedabove. The luminescence intensity arising from DPA detection can becorrelated to the concentration of bacterial spores. When this detectionmethod is coupled to an aerosol capture device that suspends aerosolizedspores into a terbium containing solution, unattended monitoring ofbacterial spores in the air is enabled.

[0070] In general, the method comprises the steps of capturingaerosolized bacterial spores with an aerosol sampler or impactor ofwhich there are many commercial models are available. The capturedspores are then lysed using microwave radiation, autoclaving, or othermethods that release DPA from the core of the spores. The released DPAthen binds terbium ions or other chromophores that give rise toluminescence turn-on upon DPA binding. The luminescence turn-on ismonitored by a luminescence spectrometer or fluorimeter. Continuoussampling of the air while monitoring for luminescence turn-on gives riseto an alarm capability for aerosolized bacterial spores, which does notrequire human participation over extended periods, such as time periodsof the order of 8 hours.

[0071] In the illustrated embodiment stock solutions of purifiedBacillis subtilis spores were purchased from Raven Biological. ALovelace nebulizer was used to generate an aerosol 40 of the bacterialspore air suspensions. The spore “smoke” detector instrument as shown inthe diagram of FIG. 7, is comprised of three components: (1) abiosampler 42 for aerosol capture, (2) a microwave with temperature andpressure control 44 for releasing the DPA from the spores, and (3) alifetime-gated luminescence spectrometer 50 for luminescence detection.The lifetime gating works by exciting the sample with a short Xe-lampflash 51 and waiting several microseconds before detecting light fromthe sample 46, thus eliminating the background fluorescence fromimpurities with 10-ns fluorescent lifetimes.

[0072] The biosampler 42, filled with 20 ml of 10 μM TbCl₃ glycerolsolution, has a 95% transfer efficiency for microbe-containing aerosols.Once bacterial spores are suspended in the biosampler collection vessel47, microwaving completely or sufficiently releases DPA into bulksolution 46 within 8 minutes or less. The resulting free DPA then bindsTb in bulk solution, giving rise to luminescence turn-on under UVexcitation. A fiber optic probe 48 immersed in the sample solutiontransmits the Luminescence to the spectrometer 50. Spectrometer 50 iscoupled to alarm circuit 52 which then generates an appropriate alarmsignal when a predetermined detection occurs, namely a wireless or wiredsignal with identification information is generated and transmitted to aremote monitoring station. The monitoring station may monitor aplurality of remote biosensors such as shown in FIG. 7 and providing acontinuous time, date, place and biomeasurement report from them.

[0073] While the biosampler 42 is continually sampling the air, a cyclecomprising an 8-minute microwaving step at 140° C. at 1 atmosphere, a 7minute cooling period, and a 30 second luminescence measurement isperformed repeatedly. Cooling down to room temperature is requiredbecause the binding constant for the Tb-DPA complex at 140° C. is muchlower than at room temperature, thus leading to near zero fraction boundat 140° C. FIG. 8a shows the time course of the luminescence intensityat 543.5 nm versus time for the online monitoring for aerosolizedbacterial spores in the device of FIG. 7. After five data points arecollected in the time interval between t=0 and 63 minutes, we initiatedthe nebulizer for 5 minutes to generate aerosolized bacterial spores,which were directed to the inlet of the biosampler 42. The sixth datapoint at t=81 min. clearly shows the presence of Tb-DPA luminescence,thus signaling the presence of bacterial spores. The luminescenceintensity in the plateau region after 130 minutes corresponds to a sporeconcentration of 10⁵ spores/ml. The luminescence increases for two moreheating and cooling cycles and then plateaus 60 minutes after theinitiation of the spore event.

[0074]FIG. 8b shows the luminescence spectra before and after thegeneration of aerosolized bacterial spores. Clearly, the signal-to-noiseratio of 10, one cycle after spore introduction, shows that we candetect aerosolized spores with a response time of about 15 minutes.Spore lysing methods, such as sonication with microbeads, that do notrequire high temperature will lead to increased sampling rates.

[0075] Thus, we have demonstrated quantification of aerosolizedbacterial spores with a response time of about 15 minutes or less, asensitivity of 10⁵ spores/ml, and a dynamic range of four orders ofmagnitude. The sensitivity can be improved by optimizing aerosolcollection and spectrometer performance. Ultimately, the most attractivefeature we have demonstrated is the unattended monitoring of aerosolizedbacterial spores for the duration of a workday (i.e.—8 hrs).

[0076] Many alterations and modifications may be made by those havingordinary skill in the art without departing from the spirit and scope ofthe invention. Therefore, it must be understood that the illustratedembodiment has been set forth only for the purposes of example and thatit should not be taken as limiting the invention as defined by thefollowing claims. For example, notwithstanding the fact that theelements of a claim are set forth below in a certain combination, itmust be expressly understood that the invention includes othercombinations of fewer, more or different elements, which are disclosedin above even when not initially claimed in such combinations.

[0077] The words used in this specification to describe the inventionand its various embodiments are to be understood not only in the senseof their commonly defined meanings, but to include by special definitionin this specification structure, material or acts beyond the scope ofthe commonly defined meanings. Thus if an element can be understood inthe context of this specification as including more than one meaning,then its use in a claim must be understood as being generic to allpossible meanings supported by the specification and by the word itself.

[0078] The definitions of the words or elements of the following claimsare, therefore, defined in this specification to include not only thecombination of elements which are literally set forth, but allequivalent structure, material or acts for performing substantially thesame function in substantially the same way to obtain substantially thesame result. In this sense it is therefore contemplated that anequivalent substitution of two or more elements may be made for any oneof the elements in the claims below or that a single element may besubstituted for two or more elements in a claim. Although elements maybe described above as acting in certain combinations and even initiallyclaimed as such, it is to be expressly understood that one or moreelements from a claimed combination can in some cases be excised fromthe combination and that the claimed combination may be directed to asubcombination or variation of a subcombination.

[0079] Insubstantial changes from the claimed subject matter as viewedby a person with ordinary skill in the art, now known or later devised,are expressly contemplated as being equivalently within the scope of theclaims. Therefore, obvious substitutions now or later known to one withordinary skill in the art are defined to be within the scope of thedefined elements.

[0080] The claims are thus to be understood to include what isspecifically illustrated and described above, what is conceptionallyequivalent, what can be obviously substituted and also what essentiallyincorporates the essential idea of the invention.

We claim:
 1. A method for lateral flow immunoassay for species-specificbacterial spore detection and quantification comprising: providing amatrix including terbium ions; releasing DPA from the bacterial spores;combining the terbium ions with the DPA in solution; and exciting thecombined terbium ions and DPA to generate a luminescence characteristicof the combined terbium ions and DPA to detect the bacterial spores. 2.The method of claim 1 where releasing DPA from the bacterial sporescomprises microwaving the spores.
 3. The method of claim 1 wherereleasing DPA from the bacterial spores comprises germinating the sporeswith L-alanine.
 4. The method of claim 1 where releasing DPA from thebacterial spores comprises sonicating the spores with microspheres. 5.The method of claim 1 where releasing DPA from the bacterial sporescomprises autoclaving the spores.
 6. The method of claim 1 whereexciting the combined terbium ions and DPA to generate a luminescencecharacteristic of the combined terbium ions and DPA comprises radiatingthe combined terbium ions and DPA with ultraviolet light.
 7. A methodfor lateral flow immunoassay for bacterial spore detection andquantification comprising: adding a sample of unknown bacterial sporesto a test strip; drawing the sample of unknown bacterial spores to afirst sample region on the test strip; selectively bindingspecies-specific antibodies to the sample when the unknown bacterialspores match the species-specific antibodies, otherwise leaving thesample unbound; releasing DPA from the bacterial spores in the boundsample; combining the terbium ions with the DPA; and exciting thecombined terbium ions and DPA to generate a luminescence characteristicof the combined terbium ions and DPA to detect the bacterial spores. 8.The method of claim 7 further comprising: adding a standard of knownbacterial spores with known concentration to the test strip; drawing thestandard of known bacterial spores to a second sample region on the teststrip; selectively binding species-specific antibodies to the standardwhen the known bacterial spores match the species-specific antibodies,otherwise leaving the standard unbound; releasing DPA from the bacterialspores in the bound standard; combining the terbium ions with the DPA;exciting the combined terbium ions and DPA to generate a luminescencecharacteristic of the combined terbium ions and DPA to detect thebacterial spores of the standard; and comparing the intensity of theexcited luminescence from the sample with the standard to derive aquantification of the spore concentration in the sample.
 9. The methodof claim 8 further comprising confirming arrival of the sample andstandard in the first and second sample regions respectively by means ofa visual indicator.
 10. The method of claim 8 where releasing DPA fromthe bacterial spores in the sample and standard comprises microwavingthe spores.
 11. The method of claim 8 where releasing DPA from thebacterial spores in the sample and standard comprises germinating thespores with L-alanine.
 12. The method of claim 8 where releasing DPAfrom the bacterial spores in the sample and standard comprisessonicating the spores with microspheres.
 13. The method of claim 8 wherereleasing DPA from the bacterial spores in the sample and standardcomprises autoclaving the spores.
 14. The method of claim 8 whereexciting the combined terbium ions and DPA in the sample and standard togenerate a luminescence characteristic of the combined terbium ions andDPA comprises radiating the combined terbium ions and DPA in the sampleand standard with ultraviolet light.
 15. A method for live/dead assayfor bacterial spores comprising: providing a solution including terbiumions in a sample of live and dead bacterial spores; releasing DPA fromviable bacterial spores by germination from a first unit of the sample;combining the terbium ions with the DPA in solution released from viablebacterial spores; and exciting the combined terbium ions and DPAreleased from viable bacterial spores to generate a first luminescencecharacteristic of the combined terbium ions and DPA to detect the viablebacterial spores; releasing DPA from dead bacterial spores in a secondunit of the sample by autoclaving, sonication or microwaving; combiningthe terbium ions with the DPA in solution released from dead bacterialspores; and exciting the combined terbium ions and DPA released fromdead bacterial spores to generate a second luminescence characteristicof the combined terbium ions and DPA to detect the dead bacterialspores; and generating a ratio of the first to second luminescence toyield a fraction of bacterial spores which are alive.
 16. A method forlifetime-gated measurements of bacterial spores to eliminate anyfluorescence background from organic chromophores comprising: providinga solution including terbium ions with a sample of bacterial spores;labeling the bacterial spore contents with a long-lifetime lumophore;releasing DPA from the bacterial spores; combining the terbium ions withthe DPA in solution; and exciting the combined terbium ions and DPA fora first time period; waiting a second time period before detectingluminescence; and detecting a luminescence characteristic of thecombined terbium ions and DPA after the second time period during adefined temporal window synchronized with luminescence timed from thelong lifetime lumophore to detect the bacterial spores.
 17. The methodof claim 16 where the first time period of excitation is of the order ofnanoseconds, where the second time period is of the order ofmicroseconds and where the defined temporal window is of the order ofmilliseconds.
 18. The method of claim 17 where the first time period ofexcitation is of the order of 1-10 nanoseconds, where the second timeperiod is of the order of tens of microseconds and where the definedtemporal window is of the order of 1-10 milliseconds.
 19. The method ofclaim 16 where the first time period of excitation is of the order ofnanoseconds, where the second time period is of the order of tenths totens of milliseconds and where the defined temporal window is of theorder of hundreds of microseconds.
 20. A method for unattendedmonitoring of bacterial spores in the air comprising: collectingbacterial spores carried in the air; suspending the collected bacterialspores in a solution including terbium ions; releasing DPA from thebacterial spores; combining the terbium ions with the DPA in solution;and exciting the combined terbium ions and DPA to generate aluminescence characteristic of the combined terbium ions and DPA;detecting the luminescence to determine the presence of the bacterialspores; and generating an alarm signal when the presence of bacterialspores is detected or the concentration thereof reaches a predeterminedmagnitude.
 21. The method of claim 20 where collecting bacterial sporescarried in the air comprises capturing the bacterial spores with anaerosol sampler or impactor.
 22. The method of claim 20 where detectingthe luminescence to determine the presence of the bacterial sporescomprises monitoring the luminescence with a spectrometer orfluorimeter.
 23. The method of claim 20 where collecting bacterialspores carried in the air comprising continuously sampling the air andwhere detecting the luminescence to determine the presence of thebacterial spores comprises continuously monitoring the luminescence. 24.The method of claim 23 where releasing DPA from the bacterial sporescomprising microwaving the bacterial spores to heat the solution andwhere combining the terbium ions with the DPA in solution comprisescooling the heated solution to increase the fraction of bound Tb-DPAcomplex.
 25. An apparatus for unattended monitoring of bacterial sporesin the air comprising: a biosampler for capturing the bacterial sporesin the air and having a collection vessel containing a solutionincluding terbium ions into which the captured bacterial spores aresuspended; means for releasing DPA from the bacterial spores in thesolution to allow the DPA to combine with the terbium ions to form aTb-DPA complex; an energy source for exciting the Tb-DPA complex togenerate luminescence; an electro-optical circuit to measure theluminescence; and an alarm circuit coupled to the electro-opticalcircuit to detect a bacterial spore concentration above a predeterminedthreshold.
 26. An apparatus for lateral flow immunoassay for bacterialspore detection and quantification comprising: a strip of material forproviding lateral capillary flow of a solution including terbium ionsacross the strip; an input region on the strip for receiving a liquidsample containing terbium ions, the first zone being provided with afirst antibody for specific binding to a specific specie of bacterialspores; a sample region of the strip laterally displaced from the inputregion and communicated thereto by means of capillary flow therebetween,the sample region being provided with a second antibody to capturebacterial spores with the attached first antibody and to immobilizethem; means for releasing DPA from the bacterial spores in the sampleregion of the strip to then allow the terbium ions to combine with theDPA in solution; and an energy source to excite the combined terbiumions and DPA in the sample region of the strip to generate aluminescence characteristic of the combined terbium ions and DPA; and aluminescence detector to identify the presence or measure theconcentration of the bacterial spores in the sample region of the strip.27. The apparatus of claim 26 where the means for releasing DPA from thebacterial spores in the sample region of the strip comprises a microwaveheater, means for adding L-alanine to the solution, means for sonicatingthe spores with microspheres, or an autoclave.